Pharmaceutically acceptable salts of thymodepressin and processes for their manufacture

ABSTRACT

The present invention relates to pharmaceutically acceptable crystalline and amorphous salts of D-isoglutamyl-D-tryptophan as well as processes for their manufacture, pharmaceutical compositions comprising them, and their uses in the preparation of pharmaceutical compositions for the treatment of various conditions and/or diseases. In particular, the present invention relates to D-isoglutamyl-D-tryptophan potassium salt (1:1), D-isoglutamyl-D-tryptophan lithium salt (1:1), D-isoglutamyl-D-tryptophan calcium salt (2:1), D-isoglutamyl-D-tryptophan magnesium salt (2:1), and D-isoglutamyl-D-tryptophan organic ammonium salts (1:1).

STATEMENT OF RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 12/520,337 (now U.S. Pat. No. 8,138,221), which entered nationalphase on Jun. 19, 2009 from International Patent Application No.PCT/CA2007/002235, filed Dec. 13, 2007, which claims the benefit ofCanadian Patent Application No. 2571645, filed Dec. 19, 2006, all ofwhich are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to novel crystalline and amorphouspharmaceutically acceptable salts of D-isoglutamyl-D-tryptophan. Inparticular, the present invention relates to D-isogl!utamyl-D-tryptophanpotassium salt (1:1), D-isoglutamyl-D-tryptophan lithium salt (1:1),D-isoglutamyl-D-tryptophan calcium salt (2:1),D-isoglutamyl-D-tryptophan magnesium salt (2:1), andD-isoglutamyl-D-tryptophan organic ammonium salts (1:1) which haveimproved properties over amorphous D-isoglutamyl-D-tryptophan,crystalline D-isoglutamyl-D-tryptophan and D-isoglutamyl-D-tryptophandisodium salt. The present invention also relates to processes for themanufacture of these novel salts of D-isoglutamyl-D-tryptophan.

BACKGROUND OF THE INVENTION

The compound D-isoglutamyl-D-tryptophan (also known as H-D-iGlu-D-Trp-OHor Thymodepressin) is a synthetic hemoregulatory dipeptide having thefollowing formula:

Thymodepressin is the free diacid having Chemical Abstracts Service(CAS) Registry Number® of 186087-26-3. U.S. Pat. No. 5,736,519 disclosesH-D-iGlu-D-Trp-OH and a process for its preparation wherein it ispurified by ion exchange chromatography. It is an immunosuppressant andselectively inhibits proliferation of hemopoietic precursor cells andstimulates granulocyte and lymphocyte apoptosis (Sapuntsova, S. G., etal. (May 2002), Bulletin of Experimental Biology and Medicine, 133(5),488-490).

Thymodepressin is currently being sold in Russia as the disodium salt ofD-isoglutamyl-D-tryptophan in liquid formulation for injection andintranasal administration for the treatment of psoriasis and atopicdermatitis. The solid form of the disodium salt ofD-isoglutamyl-D-tryptophan is an amorphous powder which is hygroscopicand very difficult to handle. The disodium salt ofD-isoglutamyl-D-tryptophan has the molecular formula C₁₆H₁₇N₃Na₂O₅ andthe following chemical structure:

which is reported in Kashirin, D. M., et al. (2000), PharmaceuticalChemistry Journal, 34(11), 619-622.

Through investigations in our laboratory, we have determined that thefreeze-dried disodium salt of D-isoglutamyl-D-tryptophan is extremelyhygroscopic turning into a gel in a matter of minutes in air and cannoteasily be handled.

A powdery or amorphous form of a compound, intended for pharmaceuticaluse may give rise to manufacturing problems due to bulk density issues,hygroscopicity and variable water content that cannot be corrected byvacuum drying. D-isoglutamyl-D-tryptophan is a dipeptide and the dryingof an amorphous form at elevated temperature, for example, 80-100° C.under vacuum is not recommended. Thus, there are serious difficultiesexperienced during the purification of the disodium salt ofD-isoglutamyl-D-tryptophan and obtaining the pure disodium salt on amanufacturing scale. Further, there is no published procedure for itspreparation.

The monosodium salt of D-isoglutamyl-D-tryptophan is identified by theCAS Registry Registry System and is listed in the CAS REGISTRY^(SM) Filewith a CAS Registry Number® of 863988-88-9. However, there are noreferences citing the substance and thus no publication of its identity,its physical and/or chemical properties, its characterization or aprocedure for its preparation. Freeze-dried powders of mono sodium anddisodium salts of peptide drugs may not have controllable powder bulkdensity ranges for formulation. They may require significant investmentin freeze-dried dispersion technology.

Therefore, there is a need to develop alternative pharmaceuticallyacceptable salts of D-isoglutamyl-D-tryptophan which are crystalline.Such crystalline salts can generally be purified more easily than anamorphous form and may possess other advantageous properties, forexample in relation to their particular crystalline form and/or theirsolubility characteristics and/or their lack of hygroscopicity and/ortheir stability characteristics, including their thermal stabilityproperties and/or their ability to undergo oxidative degradation.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a number of novel,stable and pharmaceutically acceptable salts of thymodepressin forformulation development.

Not all salts of D-isoglutamyl-D-tryptophan are chemically stable (suchas, for example, the amorphous disodium salt). However, we have inventedstable, novel metal, and organic ammonium salts ofD-isoglutamyl-D-tryptophan (H-D-i-Glu-D-Trp-OH), which provide the basisfor the present invention.

In one aspect of the present invention, there is provided novel salts ofD-isoglutamyl-D-tryptophan. These novel salt forms are compounds offormula I,

wherein M is selected from the group consisting of lithium andpotassium; formula II,

wherein B is selected from the group consisting of magnesium andcalcium; and formula III,

wherein A is selected from the group consisting of tert-butylammonium,tris(hydroxymethyl)methylammonium andmethyl-(2,3,4,5,6-pentahydroxy-hexyl)-ammonium.

Particularly, in the novel salts of formula I, the lithium or potassiumcation displaces one hydrogen atom, on the carboxylic portion of thecompound D-isoglutamyl-D-tryptophan. It has been determined in ourlaboratory that the novel salts of formula I of the present invention,wherein M is potassium (i.e. the mono potassium salt ofD-isoglutamyl-D-tryptophan), can exist in different forms, in particularin amorphous or non-crystalline form and in crystalline form. It hasalso been determined in our laboratory that the novel salts of formula Iof the present invention, wherein M is lithium (i.e. the mono lithiumsalt of D-isoglutamyl-D-tryptophan), can exist in different forms, inparticular in amorphous or non-crystalline form and in crystalline form.Therefore, the present invention relates to the mono lithium and monopotassium salts of D-isoglutamyl-D-tryptophan in any of their forms.

In another aspect of the present invention, there is provided novellithium, and potassium salts of D-isoglutamyl-D-tryptophan, the compoundof formula I.

In another aspect of the present invention, there is provided thecrystalline potassium salt of D-isoglutamyl-D-tryptophan, the compoundof formula I.

In another aspect of the present invention, there is provided thecrystalline lithium salt of D-isoglutamyl-D-tryptophan, the compound offormula I.

The present invention also relates to a crystalline form of themagnesium salt of D-isoglutamyl-D-tryptophan and a semi-crystalline ofthe calcium salt of D-isoglutamyl-D-tryptophan wherein the calcium ormagnesium cation displaces one hydrogen atom on the carboxylic portionof the compound of D-isoglutamyl-D-tryptophan. TheD-isoglutamyl-D-tryptophan calcium or magnesium salt is formed in theratio 2:1 as illustrated in formula II.

It has been determined in our laboratory that the magnesium salt of thepresent invention (i.e. the magnesium salt of thymodepressin [1:2)exists in crystalline form, while the calcium salt of the presentinvention (i.e. the calcium salt of thymodepressin [1:2) issemi-crystalline with the percent crystallinity not exceeding about 67%.

In another aspect of the present invention, there is provided the novelcrystalline magnesium salt of D-isoglutamyl-D-tryptophan, the compoundof formula II.

In another aspect of the present invention, there is provided thecalcium salt of D-isoglutamyl-D-tryptophan, the compound of formula II.

The present invention also relates to the organic amine salts of formulaIII wherein A is selected from the group consisting oftert-butylammonium, tris(hydroxymethyl)methylammonium, and methyl-(2,3,4,5,6-pentahydroxy-hexyl)-ammonium. It has been determined in ourlaboratory that these salts of formula III are amorphous.

In another aspect of the present invention, there is provided the noveltert-butylammonium, tris(hydroxymethyl)methylammonium,methyl-(2,3,4,5,6-pentahydroxy-hexyl)-ammonium salt ofD-isoglutamyl-D-tryptophan, the compound of formula III.

In another aspect of the present invention, there is provided a processfor preparing the salts of formulas I, II, and III from the dipeptideD-isoglutamyl-D-tryptophan.

In another aspect of the present invention, there is provided a processfor preparing said salts of D-isoglutamyl-D-tryptophan (the compound offormula I, and II) from salt exchange of the D-isoglutamyl-D-tryptophanammonium salt (1:1).

In an earlier patent application filed in Canada on Nov. 28, 2006, theApplicant discloses methods for the manufacturing ofD-isoglutamyl-D-tryptophan and its mono ammonium salt, a novel stablecrystalline form of D-isoglutamyl-D-tryptophan and its mono ammoniumsalt. The D-isoglutamyl-D-tryptophan and its mono ammonium salt used inthe present invention may be prepared by the process described in theaforementioned patent application.

In another aspect of the present invention, there is provided apharmaceutical composition comprising any of the novel salts describedabove and at least one pharmaceutically acceptable carrier.

The pharmaceutical composition may be prepared by combining any of thenovel salts described above and at least one pharmaceutically acceptablecarrier. In another aspect of the present invention, there is provided aprocess for making a pharmaceutical composition comprising combining anyof the novel salts described above and at least one pharmaceuticallyacceptable carrier.

In another aspect of the present invention, there is provided the use ofany of the novel salts described herein in the preparation of amedicament for treating psoriasis, in a subject in need thereof.

A further feature of the crystalline salts of the present invention isthat they can also advantageously be used as intermediates in themanufacture of the non-crystalline salt, to enable isolation ofnon-crystalline salt with a purity level and uniformity suitable forformulation to meet exacting pharmaceutical requirements andspecifications. Examples of those salts are lithium, sodium, potassiumand ammonium salt. Recrystallization techniques generally removeimpurities in its process, while the purification of amorphous peptidedrug substances requires preparative reverse phase high pressure liquidchromatography, which is not economical.

Other and further advantages and features of the present invention willbe apparent to those skilled in the art from the following detaileddescription thereof taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings:

FIG. 1A is an X-ray Powder Diffraction (XRPD) pattern of potassium saltof D-isoglutamyl-D-tryptophan (1:1).

FIG. 1B is a Fourier Transform Infrared (FTIR) spectrum of crystallinepotassium salt of D-isoglutamyl-D-tryptophan (1:1).

FIG. 1C is an FTIR spectrum of amorphous potassium salt ofD-isoglutamyl-D-tryptophan (1:1).

FIG. 2A is an XRPD pattern of Lithium Salt of D-isoglutamyl-D-tryptophan(1:1).

FIG. 2B is an FTIR spectrum of crystalline lithium salt ofD-isoglutamyl-D-tryptophan (1:1).

FIG. 2C is an FTIR spectrum of amorphous lithium salt ofD-isoglutamyl-D-tryptophan (1:1).

FIG. 3A is an XRPD pattern of Magnesium salt ofD-isoglutamyl-D-tryptophan (1:2).

FIG. 3A is an FTIR spectrum of Magnesium salt ofD-isoglutamyl-D-tryptophan (1:2).

FIG. 4A is a PXRD pattern of Calcium salt of D-isoglutamyl-D-tryptophan(1:2).

FIG. 4A is a PXRD pattern of Calcium salt of D-isoglutamyl-D-tryptophan(1:2).

FIG. 4C is an FTIR spectrum of calcium salt ofD-isoglutamyl-D-tryptophan (1:2) using material from FIG. 4A.

FIG. 5 is a speciation plot of the dipeptide D-isoglutamyl-D-tryptophanat different pHs.

FIG. 6 is an FTIR spectrum of amorphous salt of tert-butylamine andD-isoglutamyl-D-tryptophan (1:1).

FIG. 7 is an FTIR spectrum of amorphous monotris(hydroxymethyl)aminomethane salt of D-isoglutamyl-D-tryptophan(1:1).

FIG. 8 is an FTIR spectrum of amorphous mono N-methyl-D-glucamine saltof D-isoglutamyl-D-tryptophan (1:1).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

As mentioned above, the present invention relates to the novel metalsalts and organic amine salts of D-isoglutamyl-D-tryptophan.

As used herein, D-isoglutamyl-D-tryptophan is the free diacid

The chemistry of amino acids or simple dipeptides is complicated by thefact that the —NH2 group is a base and the —CO₂H group is an acid. Inaqueous solution, an H⁺ ion is therefore transferred from one end of themolecule to the other to form a zwitterion

Zwitterions are simultaneously electrically charged and electricallyneutral. They contain positive and negative charges, but the net chargeon the molecule is zero. Although the basis for salt formation is notentirely bound by theory, the iGlu amino acid unit of H-D-iGlu-D-Trp-OHexists as a zwitterion, and therefore, there is only one —CO₂H groupleft that is available for the formation of a salt when only oneequivalent of monovalent metal hydroxide, 0.5 equivalent of divalentmetal hydroxide B(OH)2 or one equivalent of organic amine is used toadjust the pH to neutral conditions. Examples of monovalent metalhydroxides are sodium hydroxide, lithium hydroxide and potassiumhydroxide. Examples of divalent metal hydroxides are calcium hydroxideand magnesium hydroxide.

When H-D-iGlu-D-Trp-OH monovalent metal salt of formula I is drawn inthe format shown above, only one CO₂H group can accommodate onemonovalent metal to give the salt of formula I. Examples of thosemonovalent salts of the present invention are potassium and lithium salt(1:1). Examples of the divalent metal salts of the present invention arethe magnesium and calcium salts. Examples of the organic amine salts ofthe present invention are the tert-butylammonium,tris(hydroxymethyl)methylammonium, andmethyl-(2,3,4,5,6-pentahydroxy-hexyl)-ammonium salts. Although the priorart literature has loosely used the term thymodepressin to representboth the free diacid of D-isoglutamyl-D-tryptophan and its disodiumsalt, within the context of the present invention, thymodepressin is thefree diacid of D-isoglutamyl-D-tryptophan with the molecular formulaC₁₆H₁₉N₃O₅, and the disodium salt is the compound with the molecularformula C₁₆H₁₇N3Na₂O₅. They are two different chemical entities havingdifferent physical and chemical properties.

As used herein, the mono lithium or potassium salt is formed by thereplacement of one carboxylic group hydrogen with the metal ion lithiumor potassium with the structure shown in formula I above. The specificstructures are shown below:

As used herein, the magnesium or calcium salt is formed by thereplacement of one carboxylic group hydrogen with the metal ionmagnesium or calcium with the structure shown in formula II above. Theterm “D-isoglutamyl-D-tryptophan calcium salt (2:1)” refers herein toCa(D-isoglutamyl-D-tryptophan)₂. Similarly, the term“D-isoglutamyl-D-tryptophan magnesium salt (2:1)” refers herein toMg(D-isoglutamyl-D-tryptophan)₂. The specific structures are shownbelow:

As used herein, the organic amine salts refer to salts from the peptideand an organic amine. For example, tert-butylamine, N-methyl-D-glucamineand tromethamine are organic amines. The organic amine salt of thepresent invention is formed by the replacement of one carboxylic grouphydrogen with an organic amine with the structure shown in formula IIIabove. For example, the organic amine salt formed from tert-butylamineand D-isoglutamyl-D-tryptophan is referred to herein asD-isoglutamyl-D-tryptophan tert-butylammonium salt (1:1). The specificstructure is shown below:

The organic amine salt of the present invention formed fromN-methyl-D-glucamine and D-isoglutamyl-D-tryptophan is referred toherein as the D-isoglutamyl-D-tryptophanmethyl-(2,3,4,5,6-pentahydroxy-hexyl)-ammonium salt (1:1). The specificstructure is shown below:

The organic amine salt of the present invention formed from tromethamineand D-isoglutamyl-D-tryptophan is referred to herein as theD-isoglutamyl-D-tryptophan tris(hydroxymethyl)methylammonium salt (1:1).The specific structure is shown below:

A pharmaceutically acceptable salt of a drug substance is one that ischemically stable and can be used in a pharmaceutical composition.Unlike simple aromatic hydrocarbons, thymodepressin is a dipeptide withmulti-functional groups. The dipeptide D-isoglutamyl-D-tryptophan has analpha amine, two carboxylic acids and an indole nitrogen within the samemolecule. An ideal salt should be one that has a solution pH close toabout 7 and in the low basic pH range. Through investigations in ourlaboratory, in the search for a novel salt to address the shortcomingsof the disodium salt of D-isoglutamyl-D-tryptophan, the speciation plot(FIG. 5) was used to determine salts with ideal solution pH andsolubility that are suitable for pharmaceutical formulations. FIG. 5 isa speciation plot of H-D-iGlu-D-Trp-OH from pH 0 to 12 usingexperimentally determined pKas. In FIG. 5, LH₂=H-D-iGlu-D-Trp-OH,LH=mono carboxylic acid salt, L=dicarboxylic acid salt, and LH₃=acidaddition salt of H-D-iGlu-D-Trp-OH. The X axis provides the pH of thesolution, whereas the Y axis reports the amount of the species presentat a particular pH. From our practical experience, we use 6 ml of waterper gm of H-D-iGlu-D-Trp-OH for isolation purposes, which corresponds toa concentration of 0.5 M solution.

According to the speciation plot calculation shown in FIG. 5, the diacidform has a pH of about 2.7 to about 3 in solution. The metal salt offormula I wherein M is potassium or lithium; the divalent salt offormula II wherein B is calcium or magnesium has a solution pH close toabout 7.

Other salts such as salts of formula IV

wherein M is as defined above, salts of formula V

wherein B is as defined above, and salts of formula VI

wherein A is as defined above, are new salts.

The disodium salt (formula IV wherein M=Na) is a less stable chemicalentity as a solid. It is extremely hygroscopic and very difficult toweigh for formulation research. In solution, as per the speciation plot(FIG. 5), the pH is above 9.0 and the solution pH must be adjusted toabout 7.0 to about 7.4 in a formulation preparation.

As mentioned above, it has been determined through investigations in ourlaboratory that the potassium salt of formula I can exist in amorphousor non-crystalline form and in crystalline form, depending on theconditions under which it is obtained, as described in more detailbelow. The present invention relates to the mono potassium salt ofthymodepressin in any of its forms.

In an embodiment of the present invention, the potassium salt of formulaI is provided in amorphous form.

In another embodiment of the present invention, the potassium salt offormula I is provided in crystalline form.

In another embodiment of the present invention, the crystallinepotassium salt of formula I exhibits an X-ray powder diffractogram,obtained at λ=1.542 Å and using a radiation source of Cu Kα, comprisingpeaks at an angle 2θ of 9.91, 14.84, 15.81, 18.97, 19.76, 24.04, 24.36,24.82, 25.48, 27.49, 27.94, 28.42, 30.82, 31.28, 31.69, 32.17, 34.35,35.81, and 36.96°.

In another embodiment of the present invention, the crystallinepotassium salt of formula I has an X-ray powder diffractogramsubstantially in accordance with that shown in FIG. 1A.

As mentioned above, it has been determined through investigations in ourlaboratory that the lithium salt of formula I can exist in amorphous ornon-crystalline form and in crystalline form, depending on theconditions under which it is obtained, as described in more detailbelow. The present invention relates to the mono lithium salt ofthymodepressin in any of its forms.

In an embodiment of the present invention, the lithium salt of formula Iis provided in amorphous form.

In another embodiment of the present invention, the lithium salt offormula I is provided in crystalline form.

In another embodiment of the present invention, the crystalline lithiumsalt of formula I exhibits an X-ray powder diffractogram, obtained atλ=1.542 A and using a radiation source of Cu Kα, comprising peaks at anangle at 2θ of 13.57, 15.53, 18.71, 20.11, 23.34, 24.1, 25.09, 27.31,27.72, 28.39, 29.31, 30.19, 31.21, 32.06, 33.05, 33.62, and 37.41°.

In another embodiment of the present invention, the crystalline lithiumsalt of formula I has an X-ray powder diffractogram substantially inaccordance with that shown in FIG. 2A.

As mentioned above, it has been determined through investigations in ourlaboratory that the magnesium salt of formula II exists in crystallineform.

In another embodiment of the present invention, the magnesium salt offormula II is provided in crystalline form.

In another embodiment of the present invention, the crystallinemagnesium salt of formula II exhibits an X-ray powder diffractogram,obtained at λ=1:542 Å and using a radiation source of Cu Kα, comprisingpeaks at an angle at 2θ of 12.2, 13.74, 14.84, 16.16, 17.96, 18.52,18.94, 19.49, 21.05, 21.56, 22.56, 23.36, 24.12, 26.27, 27.65, 28.42,29.14, 30.55, 31.77, 32.62, 33.26, 35.05, 36.34, 37.22, and 38.05°.

In another embodiment of the present invention, the crystallinemagnesium salt of formula II has an X-ray powder diffractogramsubstantially in accordance with that shown in FIG. 3A.

The percent crystallinity of calcium thymodepressin or the calcium saltof formula II according to the present invention is under about 67%,more preferably under about 50%, and most preferably under about 25%.

In another embodiment of the present invention, the calcium salt offormula II is provided with a degree of crystallinity under about 67%.

The overall crystallinity measured by means of the powder X-raydiffraction technique provides additional helpful information forpharmaceutical materials that contain some amorphous material formedduring synthetic procedure. It is also a valuable measure for control oflong-term changes in the crystalline materials. Although not related toany structural and compositional features, the measured “percentcrystallinity” may be a good indicator of the stability of a particularmaterial as a function of time. The method for determining percentcrystallinity of the compound of the present invention is described inthe example below. Representative XRPD patterns of the calcium salt ofD-isoglutamyl-D-tryptophan are shown in FIGS. 4A and 4B.

In another embodiment of the present invention, there is provided thetert-butylammonium salt of formula III as shown below:

In another embodiment of the present invention, there is provided thetris(hydroxymethyl)methylammonium salt of formula III as shown below:

In another embodiment of the present invention, there is provided themethyl-(2,3,4,5,6-pentahydroxy-hexyl)-ammonium salt of formula III asshown below:

In another embodiment of the present invention, there is provided aprocess for preparing the lithium or potassium salt of formula I,comprising: (a) reacting D-isoglutamyl-D-tryptophan in water withlithium or potassium hydroxide; (b) concentrating the solution to an oiland adding isopropanol with stirring to cause the precipitation of thesalt; recovering the precipitate thereof; and vacuum drying the productto obtain the metal salt of formula I wherein M is lithium, orpotassium.

Particularly, the metal carboxylate salt of formula I is formed fromreacting a mixture of H-D-iGlu-D-Trp-OH with slightly less than aboutone equivalent of a metal hydroxide such as potassium hydroxide orlithium hydroxide, and adjusting the pH with the same metal hydroxide toa pH of about 7.0. Solvent evaporation gave an oil, which is treatedwith isopropanol to precipitate the solid salt. The salt is isolated byconventional means and dried under vacuum to give the product of formulaI.

In another embodiment of the present invention, there is provided aprocess for preparing the calcium salt of formula II, comprising: (a)reacting D-isoglutamyl-D-tryptophan in water with calcium hydroxide; (b)concentrating the solution with stirring to cause the precipitation ofthe salt; recovering the precipitate thereof; and vacuum drying theproduct to obtain the metal salt of formula II wherein B is calcium.

Particularly, the calcium salt is prepared by mixing the dipeptideH-D-iGlu-D-Trp-OH with calcium hydroxide, preferably about 0.48-0.49equivalent of calcium hydroxide per equivalent of H-D-iGlu-D-Trp-OH atice bath temperature with stirring for several hours, preferably fromabout 2.5 to about 4 hrs to give a solution. The preferred amount ofwater is about 12.5 ml of water per gm of H-D-iGlu-D-Trp-OH. The pH ofthe solution is adjusted with saturated Ca(OH)₂ to a pH of about 6 andthe insoluble particulates are filtered. The filtrate is evaporated toabout 14 to about 16% of its original volume. Upon stirring for about 14to about 18 hrs at room temperature, a solid is formed and filtered. Thecalcium salt is dried under vacuum.

In another embodiment of the present invention, there is provided aprocess for preparing the magnesium salt of formula II, comprising: (a)reacting D-isoglutamyl-D-tryptophan with magnesium ethoxide inisopropanol; (b) concentrating the solution to give a solid; mixing thesolution with water; filtering of insoluble particulates; diluting thefiltrate with water with stirring to precipitate the product; recoveringthe precipitate thereof; and vacuum drying the salt of formula IIwherein B is magnesium.

Particularly, the magnesium salt is prepared by adding H-D-iGlu-D-Trp-OHto a mixture of magnesium ethoxide in isopropanol at ice bathtemperature, preferably about 0.48-0.49 equivalent of magnesium ethoxideis used per equivalent of H-D-iGlu-D-Trp-OH. The mixture is stirred forabout 3 to about 10 hrs, preferably about 4 to about 5 hrs. The pH ofthe solution is tested by withdrawing a sample and mixing it with a fewdrops of water. Additional magnesium ethoxide is added, preferably fromabout 0.1 to about 0.12 equivalent of magnesium ethoxide and the mixtureis stirred for about 10 to about 18 hrs, preferably for about 14 toabout 16 hrs. The pH of the solution is tested by withdrawing a sampleand mixing it with a few drops of water and the pH is at about 7.0. Thesolution is evaporated under reduced pressure to give a solid, which isdissolved in water. The insoluble particulates are filtered and thefiltrate is evaporated to give a solid. The solid is mixed with water toform a suspension, and further stirred for about 3 to about 6 hrs toobtain a precipitate. The magnesium salt is filtered and dried undervacuum.

In another embodiment of the present invention, there is provided aprocess for preparing the organic amine salt of formula III, comprising:(a) reacting D-isoglutamyl-D-tryptophan in water with an organic aminein water wherein the organic amine is tert-butylamine orN-methylglucamine or tromethamine; and (b) concentrating the solution byco-evaporating with isopropanol; adding of acetone to cause theprecipitate of the salt; recovering the precipitate thereof; and vacuumdrying the product to obtain the organic ammonium salt of formula IIIwherein A is tert-butylammonium or tris(hydroxymethyl)methylammonium ormethyl-(2,3,4,5,6-pentahydroxy-hexyl)-ammonium.

Particularly, the compound of formula III wherein the counterion is anorganic amine is prepared by mixing the organic amine withH-D-iGlu-D-Trp-OH in water at ambient temperature and stirring themixture for about 12 to about 18 hrs. The solvent is co-evaporated withisopropanol and reduced under vacuum to give a solid, which is stirredwith acetone and filtered. Examples of organic amines are selected fromthe group consisting of tris(hydroxymethyl)aminomethane,N-methyl-glucamine and tert-butylamine.

In another embodiment of the present invention, there is provided amethod of salt exchange for a D-isoglutamyl-D-tryptophan saltcomprising: (a) reacting an ammonium salt represented by the formulaVII,

in aqueous solution with about one equivalent of a metal hydroxiderepresented by MOH wherein M is potassium or lithium; and (b)evaporating the solvent to give a solid which is mixed with water andisopropanol to obtain the compound of formula I wherein M is potassiumor lithium.

In another embodiment of the present invention, there is provided amethod of salt exchange for a D-isoglutamyl-D-tryptophan saltcomprising: (a) reacting an ammonium salt represented by the formula VIIin aqueous solution with about 0.5 equivalent of a metal hydroxideB(OH)₂ wherein B is calcium or magnesium; (b) evaporation of the solventto give a solid which is mixed with water and isopropanol to obtain thecompound of formula II wherein B is calcium or magnesium.

The compound of formula I can be prepared by salt exchange. The stableammonium salt of H-D-iGlu-D-Trp-OH is used as the starting material. Thefollowing is a representative process for the preparation of a compoundof formula I. A solution of the H-D-iGlu-D-Trp-OH, ammonium salt (1:1)is mixed with a metal hydroxide in water and stirred for about 15 min toabout 2 hrs, preferably for about 15 min to about 45 min. The solvent isremoved by evaporation and the residual liquid is mixed with isopropanolto afford a precipitate. The metal hydroxide is selected from the groupconsisting of lithium hydroxide, and potassium hydroxide. The X-raypowder diffraction patterns of D-isoglutamyl-D-tryptophan potassium salt(1:1) and D-isoglutamyl-D-tryptophan lithium salt (1:1) show that theyare crystalline.

When a solution of the H-D-iGlu-D-Trp-OH, ammonium salt (1:1) is mixedwith a metal hydroxide in water and stirred for about 15 min to about 2hrs, preferably for about 15 min to about 45 min, and then freeze-dried,the material so obtained is amorphous. The amorphous forms of thesodium, potassium, or lithium salts of H-D-iGlu-D-Trp-OH can be preparedby this method.

A compound of formula II can be prepared from the exchange of theammonium salt with calcium hydroxide or magnesium hydroxide. Forexample, a suspension of H-D-iGlu-D-Trp-OH and a metal hydroxide inwater such as calcium hydroxide or magnesium hydroxide is heated toabout 50-65° C. for about 1 to about 4 hrs. The solvent is reduced byevaporation. The residual liquid is mixed with isopropanol toprecipitate the H-D-iGlu-D-Trp-OH metal salt (2:1). When the metal ismagnesium, the metal salt H-D-iGlu-D-Trp-OH magnesium (2:1) obtained iscrystalline, as confirmed by the X-ray powder diffraction pattern. Whenthe metal is calcium, the isolated H-D-iGlu-D-Trp-OH calcium (2:1) issemi-crystalline with a degree of crystallinity of less than about 67%.

¹⁴N-NMR is a useful technique for the characterization of the monoammonium salt of thymodepressin. The metal salts of formula I & IIprepared by the above method are substantially free of the ammoniumsalt, as evident by the lack of the signal for NH₄ ⁺ on ¹⁴N-NMR.

We have applied the speciation plot (FIG. 5) to compute the pH range ofthe salt forms of the dipeptide H-D-iGlu-D-Trp-OH. As shown in FIG. 5,the salt of formula I or II or III is the predominant species between apH of about 6 to about 8 and makes them ideal candidates for formulationuse or incorporation into pharmaceutical compositions for dosing. Thisis particular ideal for liquid formulation, sublingual tablets, nasaldrops and sprays.

UTILITY AND ADMINISTRATION OF THE INVENTION

The potassium, lithium, calcium, magnesium, and organic amine salts ofD-isoglutamyl-D-tryptophan of the present invention may be formulatedinto pharmaceutical compositions for administration to subjects in atherapeutically active amount and in a biologically compatible formsuitable for in vivo administration, i.e. a form of the peptides to beadministered in which any toxic effects are outweighed by thetherapeutic effects.

According to the speciation plot as shown in FIG. 5, the dominantspecies at neutral pH is the mono carboxylate form of thymodepressin,that is, the mono sodium salt of the dipetideD-isoglutamyl-D-tryptophan, if the counterion is sodium. The disodiumsalt of D-isoglutamyl-D-tryptophan is extremely hygroscopic and is verydifficult to handle for dispensing purposes.

The amorphous or crystalline form of the salts of the present inventionare ideal candidates to replace the disodium salt in the preparation ofdifferent formulations useful in the treatment of the same conditionsand/or diseases that thymodepressin is useful in treating, such as, forexample, psoriasis.

Administration of the novel crystalline and amorphous salts of thepresent invention as described herein can be via any of the acceptedmodes of administration for systemically active therapeutic medicaments.These methods include oral, parenteral and otherwise systemic, aerosolor topical forms.

Depending on the intended mode of administration, the compositions ofthe present invention may be in the form of solid, semi-solid or liquiddosage forms, such as, for example, tablets, suppositories, pills,capsules, powders, liquids, aerosols, suspensions, or the like,preferably in unit dosage forms suitable for single administration ofprecise dosages. The compositions of the present invention may includeat least one conventional pharmaceutical carrier or excipient andcrystalline thymodepressin or its pharmaceutically acceptable monoammonium salt and, in addition, may include other medicinal agents,pharmaceutical agents, carriers, adjuvants, etc.

For solid compositions, conventional non-toxic solid carriers include,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talcum, cellulose, glucose,sucrose, magnesium carbonate, and the like may be used. The activecompound as defined above may be formulated as suppositories using, forexample, polyalkylene glycols, for example, propylene glycol, as thecarrier. Liquid pharmaceutically administerable compositions can, forexample, be prepared by dissolving, dispersing, etc., an active compoundas defined above and optional pharmaceutical adjuvants in a carrier,such as, for example, water, saline, aqueous dextrose, glycerol,ethanol, and the like, to thereby form a solution or suspension. Ifdesired, the pharmaceutical composition to be administered may alsocontain minor amounts of nontoxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents and the like, for example,sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate,triethanolamine oleate, etc. Actual methods of preparing such dosageforms are known, or will be apparent, to those skilled in this art; forexample, see Remington: The Science and Practice of Pharmacy, 21^(st)Edition, 2006, Part 5, Pharmaceutical Manufacturing, Chapters 37, 39,41-47 and 50, pp. 702-719, 745-775, 802-938, and 1000-1017 (formerlyknown as Remington's Pharmaceutical Sciences), David B. Troy (Ed.),Lipincott Williams & Wilkins, Baltimore, Md. The composition orformulation to be administered will, in any event, contain a quantity ofthe active compound(s) in an amount effective to alleviate the symptomsof the subject being treated.

Parenteral administration is generally characterized by injection,either subcutaneously, intramuscularly or intravenously. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like. Inaddition, if desired, the pharmaceutical compositions to be administeredmay also contain minor amounts of non-toxic auxiliary substances such aswetting or emulsifying agents, pH buffering agents and the like, such asfor example, sodium acetate, sorbitan monolaurate, triethanolamineoleate, etc.

For the salts of the present invention, either oral or nasal (bronchial)administration is preferred, depending on the nature of the disorderbeing treated.

For oral administration, a pharmaceutically acceptable non-toxiccomposition is formed by the incorporation of any of the normallyemployed excipients, such as, for example pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium, carbonate, and the like. Suchcompositions take the form of solutions, suspensions, tablets, pills,capsules, powders, sustained release formulations and the like. Suchcompositions may contain 1%-95% active ingredient, preferably 25-70%.

Oral and nasal administration to the lungs can also be effected byaerosol delivery forms. For aerosol administration, the activeingredient is preferably supplied in finely divided form along with asurfactant and a propellant. Typical percentages of active ingredientsare from about 0.01 to about 20% by weight, preferably from about 0.04to about 1.0%.

Surfactants must, of course, be non-toxic, and preferably soluble in thepropellant. Representative of such agents are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,olestearic and oleic acids with an aliphatic polyhydric alcohol or itscyclic anhydride such as, for example, ethylene glycol, glycerol,erythritol, arabitol, mannitol, sorbitol, the hexitol anhydrides derivedfrom sorbitol (the sorbitan esters sold under the name SPANS®) and thepolyoxyethylene and polyoxypropylene derivatives of these esters. Mixedesters, such as mixed or natural glycerides may be employed. Thepreferred surface-active agents are the oleates or sorbitan, e.g., thosesold under the names ARLACEL® C (Sorbitan sesquioleate), SPAN® 80(sorbitan monooleate) and SPAN® 85 (sorbitan trioleate). The surfactantmay constitute from about 0.1 to about 20% by weight of the composition,preferably from about 0.25 to about 5%.

The balance of the composition is ordinarily propellant. Liquefiedpropellants are typically gases at ambient conditions, and are condensedunder pressure. Among suitable liquefied propellants are the loweralkanes containing up to five carbons, such as butane and propane; andpreferably fluorinated or fluorochlorinated alkanes, such as are soldunder the name FREON®. Mixtures of the above may also be employed.

In producing the aerosol, a container equipped with a suitable valve isfilled with the appropriate propellant, containing the finely dividedactive ingredient and surfactant. The ingredients are thus maintained atan elevated pressure until released by action of the valve.

For topical administration, these compositions comprise an effectiveamount of a compound of this class in admixture with a pharmaceuticallyacceptable non-toxic carrier. A suitable range of composition would befrom about 0.1% to about 10% active ingredient, and the balance carrier,preferably from about 1 to about 2% active ingredient. The concentrationof active ingredient in pharmaceutical compositions suitable for topicalapplication will vary depending upon the particular activity of thecompound used in conjunction with the condition and subject to betreated. Suitable carriers or medicament vehicles for topicalapplication of these compounds include creams, ointments, lotions,emulsions, solutions and the like.

For example, a suitable ointment for topical application of compounds ofthe invention contains from about 15 to about 45 percent of a saturatedfatty alcohol having 16 to 24 carbon atoms such as cetyl alcohol,stearyl alcohol, behenyl alcohol, and the like and from about 45 toabout 85 wt. percent of a glycol solvent such as propylene glycol,polyethylene glycol, dipropylene glycol, and mixtures thereof. Theointment may also contain from about 0 to about 15 wt. percent of aplasticizer such as polyethylene glycol, 1,2,6-hexanetriol, sorbitol,glycerol, and the like; from about 0 to about 15 wt. percent of acoupling agent such as a saturated fatty acid having from 16 to 24carbon atoms, e.g., stearic acid, palmitic acid, behenic acid, a fattyacid amide e.g., oleamide, palmitamide, stearamide, behenamide and anester of a fatty acid having from 16 to 24 carbon atoms such as sorbitolmonostearate, polyethylene glycol monostearate, polypropylene glycol orthe corresponding mono-ester of other fatty acids such as oleic acid andpalmitic acid; and from about 0 to about 20 wt. percent of a penetrantsuch as dimethyl sulfoxide or dimethylacetamide.

A therapeutically active amount of the salts of the present inventionmay vary according to factors such as disease state, age, sex, andweight of the individual. The dosage regime may be altered to providethe optimum therapeutic response. Generally, the daily regimen should bein the range of from about 1 to about 200 mg of peptide.

The following are examples of representative formulations and in no wayrestrict the scope of the pharmaceutical compositions encompassed by thepresent invention.

Ingredients Quantity per tablet mgs Active ingredient 25 lactose,spray-dried 20 Corn starch 153 magnesium stearate 2 The aboveingredients are thoroughly mixed and pressed into single scored tablets.Active ingredient 100 lactose, spray-dried 148 magnesium stearate 2 Theabove ingredients are mixed and introduced into a hard-shell gelatincapsule. Active ingredient 200 lactose 145 cornstarch 50 magnesiumstearate 5 The above ingredients are mixed intimately and pressed intosingle scored tablets. Active ingredient 108 lactose 15 cornstarch 25magnesium stearate 2 The above ingredients are mixed and introduced intoa hard-shell gelatin capsule. Active ingredient 150 Lactose 92 The aboveingredients are mixed and introduced into a hard-shell gelatin capsule.

An injectable preparation buffered to a pH of 7 is prepared having thefollowing composition:

Ingredients Active ingredient 0.2 g KH₂PO₄ 2 ml KOH (1N) q.s. to pH 7Water (distilled, sterile) q.s. to 20 ml

An injectable preparation buffered to a pH of 7 is prepared having thefollowing composition:

Ingredients Active ingredient 0.01 g Water (distilled, sterile) q.s. to1 ml NaOH (0.2N) q.s. to pH 7

An oral suspension is prepared having the following composition:

Ingredients Active ingredient 0.1 g fumaric acid 0.5 g methyl paraben2.0 g granulated sugar 0.1 g sorbitol (70% solution) 25.5 g Veegum K(Vanderbilt Co.) 12.85 g flavoring 1.0 g colorings 0.035 ml distilledwater q.s. to 100 mlTopical Formulation

Ingredients Grams Active compound 0.2-2 Span 60 2 Tween 60 2 Mineral oil5 Petrolatum 10 Methyl paraben 0.15 Propyl paraben 0.05 BHA (butylated0.01 hydroxy anisole) distilled water q.s. 100 ml

All of the above ingredients, except water, are combined and heated toabout 45 degrees C. with stirring. A sufficient quantity of water atabout 45 degrees C. is then added with vigorous stirring to emulsify theingredients, and water then added q.s. 100 g.

In the following, the present invention is explained in detail referringto Examples, but the present invention is not limited thereto by anymeans.

The present invention provides a pharmaceutical composition whichcomprises the lithium or potassium salt of formula I in any of its formsand one or more pharmaceutically acceptable excipients.

The present invention also provides a pharmaceutical composition whichcomprises the calcium or magnesium salt of formula II in any of itsforms and one or more pharmaceutically acceptable excipients.

The present invention further provides a pharmaceutical compositionwhich comprises an organic amine salt of formula III in any of its formsand one or more pharmaceutically acceptable excipients.

Further details of the preferred embodiments of the present inventionare illustrated in the following examples which are understood to benon-limiting with respect to the appended claims.

EXAMPLES Example 1 Preparation of Potassium Salt ofD-isoglutamyl-D-tryptophan (1:1) from D-isoglutamyl-D-tryptophan andPotassium Hydroxide

In a 100-mL round bottom flask equipped with a magnetic stir bar wasplaced 5 mL of potassium hydroxide solution (0.5 N). The solution wascooled to 0° C. in an ice-water bath, and solid H-D-iGlu-D-Trp-OH (1.00g, 3 mmol) was added. The mixture was stirred while the pH of thesolution was adjusted to ca. 6.0 by adding a few drops of potassiumhydroxide solution (0.5 N). The solution was filtered to remove anysolid particulates. The filtrate was evaporated to dryness at a bathtemperature of about 30° C. to afford a solid. After drying under vacuumat room temperature for overnight, the salt was obtained in quantitativeyield, with a HPLC purity (peak area percent) of 98.3%. HPLC method;Column: XTerra MS C18; 5 μm, 4.6×250 mm; Mobile phase: A=the aqueousphase: 4 mM Tris, 2 mM EDTA, pH 7.4; B=the organic phase: CH₃CN;gradient: B %: 0 min. 5%, 15 min. 55%, 30 min. 55%, 32 min. 5%, 35 min.5%; Flow rate: 1 mL/min; injection volume: 5 μL; λ: 222, 254, 282, 450nm; retention time of the product: 6.41 min. The XRPD pattern of thiscrystalline material is shown in FIG. 1A; the water content byKarl-Fischer test is 0.7%; UV (water, c=23.8 μM, λ_(max) nm): 221 (ε33270), 280 (ε 5417); MS (m/z): 372.0 [M]⁺, 334.2 [C₁₆H₂₀N₃O₅]⁺, 187.9(100%). The FT-IR (KBr) spectrum is shown in FIG. 1B.

Example 2 A. Preparation of Mono Potassium Salt ofD-isoglutamyl-D-tryptophan (1:1) from the Mono Ammonium Salt ofD-isoglutamyl-D-tryptophan (1:1)

A solution of H-D-iGlu-D-Trp-OH, mono ammonium salt (1:1), (1.66 g, 4.05mmol) and potassium hydroxide (253 mg, 4.50 mmol) in water (20 mL) wasstirred at room temperature for 15 min. The pH of the solution was about9: The reaction mixture was evaporated under reduced pressure to avolume of about 1 mL. After cooling to room temperature, isopropanol wasadded until a solid precipitated out. The resulting suspension wasstirred at room temperature for 15 min, then filtered. The solid waswashed with isopropanol (2×20 mL) and ethyl acetate (20 mL), then driedunder vacuum in an oven at 42° C. overnight. An off white solid wasobtained (1.49 g, 99% yield). The water content by Karl-Fischer test is2.5%. Analytical data (XRPD pattern, FT-IR and MS spectra) are similarto those described in Example 1.

B. Preparation of Amorphous Form of Potassium Salt ofD-isoglutamyl-D-tryptophan (1:1) from the Mono Ammonium Salt ofD-isoglutamyl-D-tryptophan (1:1)

A solution of H-D-iGlu-D-Trp-OH, mono ammonium salt (1:1), (517 mg, 1.40mmol) and potassium hydroxide (82 mg, 1.46 mmol) in water (10 mL) wasstirred at room temperature for 30 minutes. The resulting mixture wasfreeze-dried overnight. An off white solid was obtained in quantitativeyield. The XRPD pattern spectrum confirmed that this material isamorphous. ¹H NMR (D₂O) δ: 7.69 (d, J=7.9 Hz, 1H), 7.48 (d, J=8.2 Hz,1H), 7.23 (t, J=7.6 Hz, 1H), 7.22 (s, 1H), 7.16 (t, J=7.4 Hz, 1H), 4.59(dd, J=8.7, 4.8 Hz, 1H), 3.51 (dd, J=6.8, 5.8 Hz, 1H), 3.38 (dd, J=14.8,4.8 Hz, 1H), 3.11 (dd, J=14.8, 8.8 Hz, 1H), 2.20-2.49 (m, 2H) and1.85-1.94 (m, 2H); ¹³C NMR (D₂O) δ: 181.4, 177.0, 176.6, 138.8, 129.9,126.9, 124.5, 121.9, 121.4, 114.5, 113.2, 58.6, 57.0, 34.6 (CH₂), 30.2(CH₂) and 29.3 (CH₂); the water content by Karl-Fischer test is 5.4%;the FT-IR (KBr) spectrum is shown in FIG. 1C; MS (m/z): 371.7 [M]⁺,334.2 [C₁₆H₂₀N₃O₅]⁺, 187.9 (100%); HPLC purity (peak area percent):99.8%, Retention time: 5.04 min; HPLC conditions: Column Waters SymmetryC18, 3.9×150 mm, 5 μm; Mobile phase: 0.035% HClO₄, pH 2/CH₃CN, 85/15,isocratic, Flow rate: 1 mL/min; λ: 220, 254, 280 nm.

Example 3 A. Preparation of Lithium Salt of D-isoglutamyl-D-tryptophan(1:1) from Mono Ammonium Salt of D-isoglutamyl-D-tryptophan (1:1) andLithium Hydroxide Monohydrate

A solution of H-D-iGlu-D-Trp-OH, mono ammonium salt (1:1), (1.40 g, 3.80mmol) and lithium hydroxide monohydrate (159 mg, 3.80 mmol) in water (20ml) was stirred at room temperature for 20 min. The pH of the solutionwas about 9. The reaction mixture was evaporated under reduced pressureto about 2 mL of solvent. After cooling down to room temperature,isopropanol was added until a solid precipitated out. The resultingsuspension was stirred at room temperature for 20 min, then filtered.The solid was washed with isopropanol (2×20 mL) and ethyl acetate (20mL), then dried under vacuum in an oven at 42° C. for overnight. Theproduct was obtained as an off white solid in quantitative yield. TheXRPD pattern of this crystalline material is shown in FIG. 2A. The watercontent by Karl-Fischer test is 10.7%. MS (m/z): 340.1 [M+1]⁺334.3[C₁₆H₂₀N₃O₅]⁺, 187.9 (100%). The FTIR (KBr) spectrum is shown in FIG.2B.

B. A solution of H-D-iGlu-D-Trp-OH, mono ammonium salt (1:1), (480 mg,1.30 mmol) and lithium hydroxide monohydrate (57 mg, 1.36 mmol) in water(10 mL) was stirred at room temperature for 30 min. The resultingmixture was freeze-dried overnight. The product was obtained as an offwhite solid in quantitative yield. The XRPD pattern confirmed that thismaterial is amorphous. ¹H NMR (D₂O) δ: 7.69 (d, J=7.8 Hz, 1H), 7.50 (d,J=8.2 Hz, 1H), 7.23 (t, J=7.1 Hz, 1H), 7.22 (s, 1H), 7.16 (t, J=7.5 Hz,1H), 4.57 (dd, J=8.7, 4.8 Hz, 1H), 3.36-3.43 (m, overlapping t and dd,2H), 3.12 (dd, J=14.7, 8.7 Hz, 1H), 2.20-2.35 (m, 2H) and 1.78-1.92 (m,2H); ¹³C NMR (D₂O) δ: 181.4, 178.1, 176.7, 138.8, 129.9, 126.9, 124.5,121.9, 121.4, 114.5, 113.2, 58.6, 57.1, 34.7 (CH₂), 30.2 (CH₂) and 29.3(CH₂); the FT-IR (KBr) spectrum is shown in FIG. 2C; The water contentby Karl-Fischer test is 11.5%. The MS spectrum is similar to that ofExample 3A; HPLC purity peak area percent): 99.8%, Retention time: 5.10min. The HPLC conditions described in Example 2B was used.

Example 4 Preparation of Lithium Salt of D-isoglutamyl-D-tryptophan(1:1) from D-isoglutamyl-D-tryptophan and Lithium Hydroxide Monohydrate

A. In a 100 mL round bottom flask equipped with a magnetic stir bar wasdissolved lithium hydroxide monohydrate (125.8 mg, 2.99 mmol) in 10 mLof water. The solution was cooled to 0° C. using an ice bath.H-D-iGlu-D-Trp-OH (1.00 g, 3 mmol) was suspended in the solution. Thesolid slowly dissolved over 2.5 h, and a clear pale pink solution wasobtained. After an additional 30 minutes of stirring, the mixture waswarmed to room temperature. The solution was filtered and carefullyconcentrated to a volume of about 4 mL. Isopropanol (25 mL) was addedslowly until solid began to form. The solution was filtered and thesolid divided into two equal parts.

B. One part of the solid from section A was washed with isopropanol(2×15 mL). The solid was first air dried and then dried under vacuum inthe oven (35° C.) overnight. The water content by Karl-Fischer test is10.6%. The XRPD pattern and the MS and FT-IR (KBr) spectra of thiscompound are similar to those described in Example 3A.

C. The second part of the solid was washed with isopropanol (2×15 mL),then with ethyl acetate (2×10 mL). The solid was first air dried andthen dried under vacuum in the oven (35° C.) for overnight. The XRPDpattern and the FT-IR (KBr) spectrum of this compound are similar tothose described in Example 3A.

The combined material from sections B and C is 0.99 g (97.6% yield).

Example 5 Preparation of Magnesium Salt of D-isoglutamyl-D-tryptophan(1:2) from D-isoglutamyl-D-tryptophan

In a 100-mL round bottom flask equipped with a magnetic stir bar wasplaced magnesium ethoxide (Aldrich, 98%, 0.206 g, 1.76 mmol) andisopropanol (15 mL). The solution was cooled to 0° C. in an ice-waterbath, and solid H-D-iGlu-D-Trp-OH (1.20 g, 3.60 mmol) was added. Thewhite suspension was stirred at room temperature for 4 h. 2 to 3 dropsof the reaction mixture was placed in a test tube, and a few drops ofdeionized water was added. The mixture was vortexed to give a clearsolution. The pH of the solution was 4.0 to 4.5. To the reaction mixturewas added magnesium ethoxide (Aldrich, 98%, 0.050 g, 0.43 mmol). Themixture was stirred at room temperature for overnight. 2-3 drops of thewhite suspension was placed in a test tube, and a few drops of deionizedwater were added. The mixture was vortexed to give a clear solution. ThepH of the solution was about 7.0. The mixture was evaporated to drynessat a bath temperature of 30° C. to give a white solid. The residue wasdissolved in 15 mL of deionized water to give a yellow solution. Thelatter was filtered to remove any solid particulates. The filtrate wasevaporated to dryness at a bath temperature of 30° C. to give a solid.The solid was suspended in deionized water (20 mL), and the mixture wasstirred for 3 h. The solid was collected by filtration and washed withice-cold deionized water (2×6 mL). The solid was air-dried first, andthen placed in a vacuum oven at 42° C. for overnight. Thus, 0.88 g (72%yield, HPLC purity (peak area percent): 99.1%) of the product wasobtained. The HPLC method described in Example 1 was used. The retentiontime for this product is 6.39 min. The XRPD pattern spectrum of thiscrystalline material is shown in FIG. 3A. The water content byKarl-Fischer test is 12.2%. MS (m/z): 689.3 [M]⁺, 334.2 [C₁₆H₂₀N₃O₅]⁺,187.9 (100%). UV (water, c=11.7 μM, λ_(max) nm): 221 (ε 57906), 280 (ε9449). The FT-IR (KBr) spectrum is shown in FIG. 3B.

Example 6 Preparation of Magnesium Salt of D-isoglutamyl-D-tryptophan(1:2) from the Mono Ammonium Salt of D-isoglutamyl-D-tryptophan (1:1)

A suspension of D-isoglutamyl-D-tryptophan, mono ammonium salt (1:1),(1.53 g, 4.15 mmol) and magnesium hydroxide in H₂O (20 mL) was heatedbetween 55° C. to 60° C. for 3 h. The resulting yellowish suspension wasevaporated under reduced pressure to about 1-2 mL. Then isopropanol (30mL) was added. The suspension was stirred for 20 min at roomtemperature, then filtered. The solid was washed successively withisopropanol (2×20 mL) and ethyl acetate (20 mL), then dried in a vacuumoven at 42° C. for overnight. A yellowish solid was obtained (1.5 g).The water content by Karl-Fischer test is 8.8%. Analytical data (XRPDpattern and FT-IR and MS spectra) are similar to those described inExample 5.

Example 7 Preparation of the Calcium Salt of D-isoglutamyl-D-tryptophan(1:2) from D-isoglutamyl-D-tryptophan (1:1) and Calcium Hydroxide

In a 100-mL round bottom flask equipped with a magnetic stir bar wasplaced calcium hydroxide (Aldrich, 99.99%, less than 3% calciumcarbonate, 0.2603 g, 3.51 mmol) and deionized water (30 mL). The cloudysolution was cooled to 0° C. in an ice-water bath, and solidH-D-iGlu-D-Trp-OH (2.404 g, 7.2 mmol) was added. The mixture was stirredfor 2.5 h to give a clear slightly pinkish solution. The pH of thesolution was adjusted to 6.0 by adding saturated calcium hydroxidesolution. The solution was filtered to remove any solid particulates.The filtrate was divided into two equal volume (about 20 ml each):solution A and solution B.

Solution A was reduced by rotary evaporation to about 4-5 mL using awater bath set to about 30° C. It was still a clear solution. Thisconcentrated solution was stirred vigorously at room temperature for 17h to give a solid. The solid was filtered by filtration and washed withice-cold deionized water (3×6 mL). The solid was air-dried first, andthen dried in a vacuum oven at 40° C. overnight to give a solid 0.70 g(55%, HPLC purity by area %: 97.7). The HPLC method described in Example1 was used. The retention time for this product is 6:39 min. The XRPDpattern of this material is shown in FIG. 4A.

The water content by Karl-Fischer test is 5.4%. MS (m/z): 705.6 [M+1}⁺,334.2 [C₁₆H₂₀N₃O₅]⁺, 187.9 (100%). UV (water, c=10.8 μM, λ_(max) nm):221 (ε 61014), 280 (ε 9943). The FT-IR (KBr) spectrum is shown in FIG.4C.

Solution B was evaporated to dryness. Deionized water (6 mL) was addedand the mixture was stirred for 16 h. The insoluble solid was filteredand dried under high vacuum at 35° C. for 48 h (0.53 g). The XRPDpattern is similar to that reported in FIG. 4A.

Example 8 Preparation of the Calcium Salt of D-isoglutamyl-D-tryptophan(1:2) from Mono Ammonium Salt of D-isoglutamyl-D-tryptophan (1:1) andCalcium Hydroxide

A suspension of mono ammonium salt (1:1) of D-isoglutamyl-D-tryptophan,(1.49 g, 4.06 mmol) and calcium hydroxide (150 mg, 2.03 mmol) in water(20 mL) was heated between 55° C. to 60° C. for 1 h. The resultingsolution was evaporated under reduced pressure to about 1-2 mL.Isopropanol (30 mL) was added. The suspension was stirred for 20 min atroom temperature, then filtered. The solid was washed successively withisopropanol (2×20 mL) and ethyl acetate (20 mL), then dried in oven at42° C. overnight. An off white solid was obtained (1.45 g). The XRPDpattern of this semi-crystalline material is shown in FIG. 4B. Thismaterial has a lower degree of crystallinity than that isolated inExample 7. The water content by Karl-Fischer test is 6.2%. MS (m/z):705.4 [M+1]⁺, 334.2 [C₁₆H₂₀N₃O₅]⁺, 187.9 (100%).

Example 9 Percent Crystallinity of Calcium Salt of D-iGlu-D-Trp by Meansof X-Ray Powder Diffraction Method

The overall crystallinity measured by means of the XRPD techniqueprovides additional helpful information for pharmaceutical materialsthat contain some amorphous material formed during synthetic procedure.It is also a valuable measure for control of long-term changes in thecrystalline materials. Although not related to any structural andcompositional features, the measured “percent crystallinity” may be agood indicator for the stability of a particular material as a functionof time.

The percent crystallinity is commonly measured as a ratio between thediffraction portion from the crystalline part of the sample, Ic, and thetotal diffraction from the same sample, I_(C+B). The values of I_(C) canbe obtained after an appropriate subtraction of the scattering portionfrom the background, I_(B).

For such kind of analyses, the diffraction is measured as total areaunder the profile of:

-   -   the whole pattern as it is collected (one may correct for the        air scattering) −I_(TOTAL)*    -   the peaks only (I_(C)) after subtraction of the background        (correction for the air scattering),    -   the background only with correction for air scattering −(I_(B)).

${C,{\% = {\frac{I_{C}}{I_{total}} \cdot 100}},{{{where}\text{:}\mspace{14mu} I_{C + B}} = {I_{C} + I_{B}}}}\mspace{31mu}$

It is worthy to note that this measurement is not recommended to bestandardized—it is very difficult (almost impossible) forinter-laboratory results to be compared. Every diffractometer has itsown scale of displaying the background level and the peak heights andareas depending on many instrumental and sample preparation factors.

The above approach is possible mostly for “point-detector”diffractometers in which the detector is moving with synchronised twicegreater speed than the sample in order to ensure the theta/2thetaconstant ratio.

However, in this study, due to the technical problems with the D5000point-detector diffraction system, all the samples were analyzed on a D8system, equipped with an area, 2D detector, and the above-mentionedapproach could not be applied. Therefore, another technique has beendeveloped for measuring the crystallinity of samples analyzed on suchsystem. Only a narrow part of the 2D diffraction image is chosen with asingle peak at the centre and a neighbour area where the background willbe measured.

Samples prepared in Example 8 have % crystallinity of around 18-20%,while samples prepared by the method of Example 7 have a % crystallinityrange between 25 to 50%.

The percent crystallinity of a sample can be improved by taking a sampleand dissolving it in water. The insoluble particulate is filtered andthe solid is allowed to slowly precipitate out of solution.

All samples were measured for calculation of their crystallinity using anarrow range of 21°-25° with a strongest reflection at 13°. Thebackground was subtracted as linear, and an empirical correlationcoefficient of 2.5 was applied.

As was pointed out above, the results thus obtained for crystallinity ofthe semi-crystalline samples should not be considered as absolute ones.None of samples from this structural type possess crystalline order highenough to be considered as absolute reference standard. Examples 7 and 8produce calcium salt of thymodepressin (1:2) with various percentcrystallinity.

Example 10 Preparation of Mono tert-butylamine Salt ofD-isoglutamyl-D-tryptophan (1:1) from D-isoglutamyl-D-tryptophan andtert-butylamine

A. To a suspension of D-iGlu-D-Trp (1.00 g, 3.00 mmol) in 25 mL ofdeionized water was added 0.7 mL (2.22 equiv) of tert-butylamine at roomtemperature (RT). The reaction mixture was clear and the pH of thesolution was about 9. After stirring at RT for 1 h, isopropanol wasadded, and volatile materials were removed in vacuo. The residual solidwas suspended in acetone, and the solid was collected by suctionfiltration. The solid was dried under vacuum at 40° C. for overnight toafford 1.16 g (95% yield) of the mono amine salt. ¹H NMR confirmed thatthe product is a mono addition salt. The XRPD pattern confirmed thatthis material is amorphous. ¹H NMR (D₂O) δ: 7.71 (d, J=8.6 Hz, 1H), 7.39(d, J=8.1 Hz, 1H), 7.13-7.16 (m, 2H), 7.07 (t, J=7.6 Hz, 1H), 4.48 (dd,J=8.3, 4.9 Hz, 1H), 3.44 (t, J=6.4 Hz, 1H), 3.28 (dd, J=14.8, 4.7 Hz,1H), 3.02 (dd, J=14.7, 8.7 Hz, 1H), 2.18-2.26 (m, 2H), 1.76-1.97 (m, 2H)and 1.26 (s, 9H). ¹⁴N NMR (D₂O) δ (ppm): 40.2 (br) and 56.3 (s), *NH₄NO₃was used as external reference with the reference signal set at 20.689ppm. The water content by Karl-Fischer test is 4.0%. MS (m/z): 407.3[M+1]⁺ (weak), 334.2 [C₁₆H₂₀N₃O₅]⁺, 187.9 (100%). The IR spectrum isshown in FIG. 6. UV (water, c=34.8 μM, λ_(max) nm): 220 (ε 31067), 280(ε 5112).

B. To a suspension of D-iGlu-D-Trp (1.00 g, 3.00 mmol) in 25 mL ofdeionized water was added 0.31 mL (1.0 equiv) of tert-butylamine at roomtemperature (RT). The reaction mixture was clear and the pH of thesolution was about 9. After stirring at RT for 1 h, isopropanol wasadded, and volatile materials were removed in vacuo. The residual solidwas suspended in acetone, and the solid was collected by suctionfiltration. The solid was dried under vacuum at 40° C. for overnight toafford 1.16 g (95% yield) of the amine salt. The analytical dataobtained for this compound (XRPD, ¹H NMR, MS, FT-IR) are similar tothose described in Example 10A above.

Example 11 Preparation of mono tris(hydroxymethyl)aminomethane salt ofD-isoglutamyl-D-tryptophan (1:1) from D-isoglutamyl-D-tryptophan andtris(hydroxymethyl)aminomethane (TRIS)

To a suspension of D-iGlu-D-Trp (1.00 g, 3.00 mmol) in 20 mL ofdeionized water was added a solution of 363 mg (1.0 equiv) oftris(hydroxymethyl)aminomethane (TRIS) in 15 ml. of deionized water atRT. The reaction mixture was clear and the pH of the solution was about7. After stirring at RT for overnight, isopropanol was added, andvolatile materials were removed in vacuo. Attempts to recrystallize thecompound using a mixture of isopropanol/water or methanol/diethyl etherfailed. The residual solid was suspended in acetone and stirred at RTfor 1 h, and the solid was collected by suction filtration. The solidwas dried under vacuum at 40° C. for overnight to afford 1.33 g of theproduct (97.5% yield). ¹H NMR (D₂O) δ: 7.61 (d, J=7.9 Hz, 1H), 7.41 (d,J=8.1 Hz, 1H), 7.14-7.17 (m, 2H), 7.08 (t, J=7.4 Hz, 1H), 4.48 (dd,J=8.5, 4.8 Hz, 1H), 3.64 (s, 6H), 3.46 (t, J=6.0 Hz, 1H), 3.28 (dd,J=14.8, 4.7 Hz, 1H), 3.02 (dd, J=14.7, 8.7 Hz, 1H), 2.17-2.28 (m, 2H)and 1.74-1.90 (m, 2H). The water content by Karl-Fischer test is 3.3%.MS (m/z): 454.9 [M+1]⁺ (weak), 334.0 [C₁₆H₂₀N₃O₅]⁺, 187.9 (100%). The IRspectrum is shown in FIG. 7; UV (water, c=36.4 μM, λ_(max) nm): 220 (ε28373), 280 (ε 4537).

Example 12 Preparation of Mono N-methyl-D-glucamine Salt ofD-isoglutamyl-D-tryptophan (1:1) from D-isoglutamyl-D-tryptophan andN-methyl-D-glucamine

To a suspension of D-iGlu-D-Trp (1.00 g, 3 mmol) in 20 mL of deionizedwater was added a solution of 586 mg (1.0 equiv) of N-methyl-D-glucaminein 15 mL of deionized water at RT. The reaction mixture was stirred forover the weekend at RT. The reaction mixture was clear and the pH of thesolution was about 7. Isopropanol was added, and volatile materials wereremoved in vacuo. The residual solid was suspended in acetone and thesolid was collected by suction filtration. The solid was dried undervacuum at 40° C. for overnight to afford the product in quantitativeyield. The XRPD pattern confirmed that this material is amorphous. ¹HNMR (D₂O) δ: 7.61 (d, J=7.9 Hz, 1H), 7.41 (d, J=8.1 Hz, 1H), 7.15-7.18(m, 2H), 7.08 (t, J=7.5 Hz, 1H), 4.47 (dd, J=8.6, 4.8 Hz, 1H), 3.99-4.02(m, 1H), 3.70-3.75 (m, 2H), 3.65-3.68 (m, 1H), 3.54-3.60 (m, 2H), 3.45(t, J=6.2 Hz, 1H), 3.27 (dd, J=14.8, 4.7 Hz, 1H), 3.02-3.13 (m, 3H),2.68 (s, 3H), 2.19-2.26 (m, 2H) and 1.75-1.95 (m, 2H). 14N NMR (D₂O) δ(ppm): 29.6 and 39.2 (br. overlapping), *NH₄N0₃ was used as externalreference with the reference signal set at 20.689 ppm. The water contentby Karl-Fischer test is 3.1%. MS (m/z): 529.5 [M+1]⁺, 334.2[C16H20N3O5I⁺, 187.9 (100%). The IR spectrum is shown in FIG. 8. UV(water, c=41.2 μM, λ_(max) nm): 220 (ε 27341), 280 (ε 4419).

Example 13 Representative Procedure for the Preparation ofD-Isoglutamyl-D-Tryptophan, Mono Ammonium Salt (1:1) fromH-D-iGlu-D-Trp-OH

H-D-iGlu-D-Trp-OH (1 g) was mixed with ammonium hydroxide (0.55M, 6 mL).The mixture was stirred and the pH was measured to be around 4.5.Ammonium hydroxide (0.55M) was added dropwise until the pH of thesolution reached between 7.0 to 7.5. Volatile materials were removed invacuo, and the residual oil was mixed with isopropanol. A whiteprecipitate appeared. After 2 h, the solid ammonium salt was collectedby suction filtration. The solid was dried to constant weight (1 g)under high vacuum for 12 h to give the D-isoglutamyl-D-tryptophan,ammonium salt (1:1).

Although preferred embodiments of the invention have been describedherein, it will be understood by those skilled in the art thatvariations may be made thereto without departing from the spirit of theinvention or the scope of the appended claims.

The invention claimed is:
 1. A process for the preparation of a metalsalt of D-isoglutamyl-D-tryptophan of formula I

wherein M is potassium, said process comprising: (a) reactingD-isoglutamyl-D-tryptophan in water with potassium hydroxide therebyforming a solution; (b) concentrating the solution to an oil; (c) addingisopropanol with stirring to the oil to cause precipitation of the saltthereby forming a precipitate; (d) recovering the precipitate; and (e)vacuum drying the precipitate to obtain the potassium salt of formula Ior (f) reacting an ammonium salt represented by formula VII

in an aqueous solution with one equivalent of potassium hydroxide; (g)evaporating the aqueous solution to give a solid; and (h) mixing thesolid with water and isopropanol to obtain the potassium salt of formulaI.
 2. A process for the preparation of a metal salt ofD-isoglutamyl-D-tryptophan of formula II,

wherein B is magnesium, said process comprising: (a) reactingD-isoglutamyl-D-tryptophan with magnesium ethoxide in isopropanolthereby forming a solution; (b) concentrating the solution to give asolid; (c) mixing the solid with water; (d) filtering insolubleparticulates thereby forming a filtrate; (e) diluting the filtrate withwater with stirring to precipitate a product; recovering the product;and (g) vacuum drying the product to obtain the magnesium salt offormula II or (h) reacting an ammonium salt represented by formula VII

in an aqueous solution with about 0.5 equivalent of a metal hydroxideB(OH)₂ wherein B is magnesium; (i) evaporating the aqueous solution togive a solid; and (j) mixing the solid with water and isopropanol toobtain the magnsium magnesium salt of formula II.
 3. A process accordingto claim 1, said process comprising: (f) reacting an ammonium saltrepresented by formula VII

in an aqueous solution with one equivalent of potassium hydroxide; (g)evaporating the aqueous solution to give a solid; and (h) mixing thesolid with water and isopropanol to obtain the potassium salt of formulaI.
 4. A process according to claim 2, said process comprising: (h)reacting an ammonium salt represented by formula VII

in an aqueous solution with about 0.5 equivalent of a metal hydroxideB(OH)₂ wherein B is magnesium; (i) evaporating the aqueous solution togive a solid; and (j) mixing the solid with water and isopropanol toobtain the magnesium salt of formula II.
 5. A process according to claim1, said process comprising: (a) reacting D-isoglutamyl-D-tryptophan inwater with potassium hydroxide thereby forming a solution; (b)concentrating the solution to an oil; (c) adding isopropanol withstirring to the oil to cause precipitation of the salt thereby forming aprecipitate; (d) recovering the precipitate; and (e) vacuum drying theprecipitate to obtain the potassium salt of formula I.
 6. A processaccording to claim 2, said process comprising: (a) reactingD-isoglutamyl-D-tryptophan with magnesium ethoxide in isopropanolthereby forming a solution; (b) concentrating the solution to give asolid; (c) mixing the solid with water; (d) filtering insolubleparticulates thereby forming a filtrate; (e) diluting the filtrate withwater with stirring to precipitate a product; (f) recovering theproduct; and (g) vacuum drying the product to obtain the magnesium saltof formula II.